A cell always requires the uptake of an amino acid as a nutrition, and such a function is exerted by an amino acid transporter which is a membrane protein present in a cell membrane. The amino acid transporter is located in a certain position in each tissue in a multicellular organism and plays an important role in the expression of a specific function of each tissue.
A transport system asc is an amino acid transport system which transports small-sized neutral amino acids such as alanine, serine and cysteine, and was reported originally with regard to an erythrocyte membrane. Thereafter, it was identified also in a cultured cell [Christensen, Physiol. Rev. Vol. 70, page 43, 1990]. The transport system asc is a transporter which is independent of sodium, i.e., whose function requires no sodium ion. Its transport substrate selectivity and transport profile are known to vary somewhat depending on cells and animal species.
While the transport system asc exhibits a high affinity to a transport substrate such as alanine, serine and cysteine, an analogous transport system is known to exist which is a transport system C whose transport substrates are also small-sized neutral amino acids such as alanine, serine and cysteine but which exhibits a lower affinity to the transport substrate [Young et al., Biochem. J. Vol. 154, page 43, 1976; Young et al., Biochem. J. Vol. 162, page 33, 1977]. The transport system C is considered to be a subclass of the transport system asc. A sheep having a genetic defect of the transport system C was identified, and its erythrocyte was found to have a reduced glutathion content, revealing the importance of the cysteine uptake via a cell membrane in the glutathion production [Young et al., Nature, Vol. 254, page 156, 1975].
However, a conventional method involves a difficulty in analyzing the details of the transport of an amino acid or its analogue via the amino acid transport system asc and the in vivo functional roles, and it has been desired to enable a detailed functional analysis by isolating a gene of a neutral amino acid transporter responsible for the function of the amino acid transport system asc.
As small-sized neutral amino acid transporters, ASCT1 and ASCT2 have been cloned [Kanai, Curr. Opin. Cell Biol., Vol. 9, page 565, 1997]. Nevertheless, they are sodium-dependent transporters, and are different in principle from the sodium-independent amino acid transport system asc. Further, a glycine transporter and a proline transporter have also been cloned, however, each transports only glycine or proline in a sodium-dependent manner, unlike to the transport system asc [Amara and Kuhar, Annu. Rev. Neurosci., Vol. 16, page 73, 1993].
The cDNAs of rBAT and 4F2hc, i.e., type II membrane glycoproteins each having only a single membrane-spanning structure which are not the transporters themselves but are considered to be amino acid transporter-activating factors, have been cloned, and are known to activate the uptake of basic amino acids together with neutral amino acids when being expressed in an oocyte of a xenopus [Palacin, J. Exp. Biol., Vol. 196, 123, 1994].
As a transporter which transports neutral amino acids selectively, neutral amino acid transporters corresponding to the transport system L, i.e., LAT1 [Kanai et al., J. Biol., Chem., Vo. 273, page 23629 to 23632, 1998] and LAT2 [Segawa et al., J. Biol. Chem, Vol. 274, page 19745 to 19751, 1999] have been cloned. It was also revealed that the LAT1 and the LAT2 are capable of exerting their functions only when being coexisting with a cofactor 4F2hc which is a single-membrane-spanning type protein. The both are independent of Na+, and the LAT1 exhibits an exchange transport activity serving to transport large-sized neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tyrosine, triptophan, methionine and histidine, while the LAT2 exhibits a wide substrate selectivity serving to transport small-sized neutral amino acids such as glycine, alanine, serine, cysteine and threonine in addition to the large-sized neutral amino acids. Nevertheless, their substrate selectivity is also different from that of the amino acid transport system asc.
As proteins analogous to the neutral amino acid transporters LAT1 and LAT2, y+LAT1 and y+LAT2 having the functions of a transport system y+L which transports neutral amino acids and basic amino acids have been cloned [Torrents et al., J. Biol. Chem., Vol. 273, page 32437 to 32445, 1998]. It was also revealed that both of y+LAT1 and y+LAT2 are capable of exerting their functions only when being coexisting with a cofactor 4F2hc. The y+LAT1 and y+LAT2 transport mainly glutamine, leucine and isoleucine as neutral amino acids, exhibiting the substrate selectivity different from that of the amino acid transport system asc.
As a transporter requiring the cofactor 4F2hc for exerting its function, xCT which is a protein analogous to the neutral amino acid transporters LAT1 and LAT2 has been cloned [Sato et al., J. Biol. Chem., 274: 11455-11458, 1999]. The xCT transports cystine and glutamic acid, exhibiting the substrate selectivity different from that of the amino acid transport system asc.
Further, as a transporter requiring another cofactor rBAT having a structure analogous to that of 4F2hc, BAT1 which is a protein analogous to the neutral amino acid transporters LAT1 and LAT2 has been cloned [Chairoungdua et al., J. Biol. Chem. 274: 28845-28848, 1999]. The BAT1 transports cystine, neutral amino acids and basic amino acids, exhibiting the substrate selectivity different from that of the amino acid transport system asc.
As described above, a molecular entity of a transporter which functions as a result of the binding to the 4F2hc and the rBAT was characterized and it was revealed that there is a group of the transporters exerting the transporting ability by forming a molecular complex with a type II glycoprotein.
Moreover, as a transporter requiring the cofactor 4F2hc for expressing a function, Asc-1 which is a protein analogous to the neutral amino acid transporters LAT1 and LAT2 has been cloned [Fukasawa et al., Biol. Chem. 275: 9690-9698, 2000]. The Asc-1 transports alanine, serine, cysteine, threonine, glycine and the like selectively, exhibits the substrate selectivity of the amino acid transport system asc, and was proven to be the first isoform of the transporting system asc.
The Asc-1 exhibits a property different from that of a traditional transporting system asc reported with regard to the erythrocyte membrane, since it transports not only L-amino acids but also D-forms of alanine, serine, cysteine and threonine, α-aminoisobutyric acid and β-alanine. Accordingly, it was believed that there is a transporter, other than the Asc-1, corresponding to the traditional transporting system asc.
In an attempt to prepare a fusion protein of a transporter with another protein, b0.+AT was combined with a single-membrane-spanning type cofactor, rBAT, for its transfer to a cell membrane [Pfeiffer et al., Mol. Biol. Cell. 10: 4135-4147, 1999]. Nevertheless, this fusion protein is the one prepared in accordance with an authentic combination of the b0.+AT and the rBAT, and is not intended to allow a transporter protein, which can not be expressed in the cell membrane because of the absence of cofactor, to be expressed forcibly on the cell membrane for the purpose of identifying its functions.